Chemokine α-5

ABSTRACT

The present invention relates to a novel CKα-5 protein which is a member of the alpha chemokine family. In particular, isolated nucleic acid molecules are provided encoding the human CKα-5 protein. CKα-5 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of CKα-5 activity. Also provided are diagnostic methods for detecting immune system-related disorders and therapeutic methods for treating immune system-related disorders.

This application is a divisional of and claims priority under 35 U.S.C.§ 120 to U.S. application Ser. No. 09/195,106, filed Nov. 18, 1998 (nowU.S. Pat. No. 6,395,514, issued May 28, 2002), which is anon-provisional of and claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 60/066,369, filed Nov. 21, 1997, which ishereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a novel human gene encoding apolypeptide which is a member of the chemokine family. Morespecifically, isolated nucleic acid molecules are provided encoding ahuman polypeptide named Chemokine Alpha-5, hereinafter referred to as“CKα-5”. CKα-5 polypeptides are also provided, as are vectors, hostcells and recombinant methods for producing the same. Also provided arediagnostic methods for detecting disorders related to the immune system,and therapeutic methods for treating such disorders. The inventionfurther relates to screening methods for identifying agonists andantagonists of CKα-5 activity.

BACKGROUND OF THE INVENTION

The ability to control the migration and “trafficking” of various celltypes is controlled by a subset of factors, or proteins, among whichchemokines are an example.

Chemokines, also referred to as intercrine cytokines, are a subfamily ofstructurally and functionally related chemotactic cytokines. Thesemolecules are usually 8–10 kd in size, however, larger membrane boundchemokine polypeptides have been identified. In general, chemokinesexhibit 20% to 75% homology at the amino acid level and arecharacterized by four conserved cysteine residues that form twodisulfide bonds. Based on the arrangement of the first two cysteineresidues, chemokines have been classified into two subfamilies, alphaand beta. In the alpha subfamily, the first two cysteines are separatedby one amino acid and hence are referred to as the “C—X—C” subfamily. Inthe beta subfamily, the two cysteines are in an adjacent position andare, therefore, referred to as the “C—C” subfamily. Thus far, at leastsixteen different members of this family have been identified in humans.

The intercrine cytokines exhibit a wide variety of functions. A hallmarkfeature is their ability to elicit chemotactic migration of distinctcell types, including monocytes, neutrophils, T lymphocytes, basophilsand fibroblasts. Many chemokines have proinflammatory activity and areinvolved in multiple steps during an inflammatory reaction. Theseactivities include stimulation of histamine release, lysosomal enzymeand leukotriene release, increased adherence of target immune cells toendothelial cells, enhanced binding of complement proteins, inducedexpression of granulocyte adhesion molecules and complement receptors,and respiratory burst. In addition to their involvement in inflammation,certain chemokines have been shown to exhibit other activities. Forexample, macrophage inflammatory protein 1 (MIP-1) is able to suppresshematopoietic stem cell proliferation, platelet factor-4 (PF-4) is apotent inhibitor of endothelial cell growth, Interleukin-8 (IL-8)promotes proliferation of keratinocytes, and GRO is an autocrine growthfactor for melanoma cells.

In light of the diverse biological activities, it is not surprising thatchemokines have been implicated in a number of physiological and diseaseconditions, including lymphocyte trafficking, wound healing,hematopoietic regulation and immunological disorders such as allergy,asthma and arthritis.

Members of the “C—C” branch exert their effects on the following cells:eosinophils which destroy parasites to lessen parasitic infection andcause chronic inflammation in the airways of the respiratory system;macrophages which suppress tumor formation in vertebrates; and basophilswhich release histamine which plays a role in allergic inflammation.However, members of one branch may exert an effect on cells which arenormally responsive to the other branch of chemokines and, therefore, noprecise role can be attached to the members of the branches.

While members of the C—C branch act predominantly on mononuclear cellsand members of the C—X—C branch act predominantly on neutrophils adistinct chemoattractant property cannot be assigned to a chemokinebased on this guideline. Some chemokines from one family showcharacteristics of the other.

The polypeptide of the present invention has the conserved cysteineresidues of the “C—X—C” region, and has amino acid sequence homology toknown chemokines.

Thus, there is a need for polypeptides that function as regulators ofthe migration of distinct cell types and of their roles in dysfunctionand disease, since disturbances of such regulation may be involved indisorders relating to hemostasis, angiogenesis, tumor metastasis,cellular migration and ovulation, as well as neurogenesis. Therefore,there is a need for identification and characterization of such humanpolypeptides which can play a role in detecting, preventing,ameliorating or correcting such disorders.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of the CKα-5polypeptide having the complete amino acid sequence shown in SEQ ID NO:2or the complete amino acid sequence encoded by a cDNA clone deposited asplasmid DNA as ATCC® Deposit Number 209231 on Aug. 29, 1997. The ATCC®is located at 10801 University Boulevard, Manassas, Va. 20110-2209. Thenucleotide sequence determined by sequencing the deposited CKα-5 clone,which is shown in FIG. 1 (SEQ ID NO:1), contains an open reading frameencoding a complete polypeptide of 254 amino acid residues, including aninitiation codon encoding an N terminal methionine at nucleotidepositions 542–544. Nucleic acid molecules of the invention include thoseencoding the complete amino acid sequence excepting the N-terminalmethionine shown in SEQ ID NO:2, or the complete amino acid sequenceexcepting the N-terminal methionine encoded by a cDNA clone in ATCC®Deposit Number 209231, which molecules also can encode additional aminoacids fused to the N-terminus of the CKα-5 amino acid sequence.

The polypeptide of the present invention has amino acid sequencehomology to known chemokines, including the conserved C—X—C cysteinepattern characteristic of the alpha subfamily of chemokines beginningwith the first cysteine from the amino terminus in SEQ ID NO:2.

CKα-5 also lacks the ELR motif found in some alpha chemokinesimmediately preceding the first cysteine residue, which is known to berequired for the neutrophil and endothelial cell chemotactic activity aswell as the angiogenic activity of IL-8.

The encoded polypeptide has a predicted leader sequence of 27 aminoacids underlined in FIG. 1; and the amino acid sequence of the predictedmature CKα-5 protein is also shown in FIG. 1 as amino acid residues28–254 and as residues 28–254 in SEQ ID NO:2. Further, the polypeptideis predicted to consist of an extracellular domain having the sequencefrom residues 28 to 205 in FIG. 1 (28 to 205 in SEQ ID NO:2), atransmembrane domain having a sequence from residue 206 to 227 in FIG. 1(206 to 227 in SEQ ID NO:2), and an intracellular domain having asequence from residue 228 to 254 in FIG. 1 (228 to 254 in SEQ ID NO:2).

Thus, one aspect of the invention provides an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence encoding the CKα-5 polypeptide having thecomplete amino acid sequence in FIG. 1 (SEQ ID NO:2); (b) a nucleotidesequence encoding the CKα-5 polypeptide having the complete amino acidsequence in FIG. 1 (SEQ ID NO:2) excepting the N-terminal methionine(i.e., positions 2 to 254 of SEQ ID NO:2); (c) a nucleotide sequenceencoding the predicted mature CKα-5 polypeptide having the amino acidsequence at positions 28–254 in FIG. 1 (28–254 in SEQ ID NO:2); (d) anucleotide sequence encoding the predicted extracellular domain of theCKα-5 polypeptide having the amino acid sequence at positions 28–205 inFIG. 1 (28–205 in SEQ ID NO:2); (e) a nucleotide sequence encoding theCKα-5 polypeptide having the complete amino acid sequence encoded by thecDNA clone contained in ATCC® Deposit No. 209231; (f) a nucleotidesequence encoding the CKα-5 polypeptide having the complete amino acidsequence excepting the N terminal methionine encoded by the cDNA clonecontained in ATCC® Deposit No. 209231; (g) a nucleotide sequenceencoding the mature CKα-5 polypeptide encoded by the cDNA clonecontained in ATCC® Deposit No. 209231; (h) a nucleotide sequenceencoding the extracellular domain of CKα-5 encoded by the cDNA clonecontained in ATCC® Deposit No. 209231; and (i) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g) or (h) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h), or (i) above, or a polynucleotide which hybridizesunder stringent hybridization conditions to a polynucleotide in (a),(b), (c), (d), (e), (f), (g), (h) or (i), above. This polynucleotidewhich hybridizes does not hybridize under stringent hybridizationconditions to a polynucleotide having a nucleotide sequence consistingof only A residues or of only T residues. An additional nucleic acidembodiment of the invention relates to an isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a CKα-5 polypeptide having an amino acidsequence in (a), (b), (c), (d), (e), (f), (g) or (h), above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofCKα-5 polypeptides or peptides by recombinant techniques.

The invention further provides an isolated CKα-5 polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of the full-length CKα-5 polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:2 or the complete aminoacid sequence encoded by the cDNA clone contained in the ATCC® DepositNo. 209231; (b) the amino acid sequence of the full-length CKα-5polypeptide having the complete amino acid sequence shown in SEQ ID NO:2excepting the N-terminal methionine (i.e., positions 2 to 228 of SEQ IDNO:2) or the complete amino acid sequence excepting the N terminalmethionine encoded by the cDNA clone contained in the ATCC® Deposit No.209231; (c) the amino acid sequence of the mature CKα-5 polypeptidehaving the amino acid sequence shown in SEQ ID NO:2 as residues 28–228or the amino acid sequence of the mature CKα-5 encoded by the cDNA clonecontained in the ATCC® Deposit No. 209231; and (d) the amino acidsequence of the extracellular domain of the CKα-5 polypeptide having theamino acid sequence shown in SEQ ID NO:2 as residues 28–205 or the aminoacid sequence of the extracellular domain of CKα-5 encoded by the cDNAclone contained in the ATCC® Deposit No. 209231. The polypeptides of thepresent invention also include polypeptides having an amino acidsequence at least 80% identical, more preferably at least 90% identical,and still more preferably 95%, 96%, 97%, 98% or 99% identical to thosedescribed in (a), (b), (c) or (d) above, as well as polypeptides havingan amino acid sequence with at least 90% similarity, and more preferablyat least 95% similarity, to those above. Polynucleotides encodingpolypeptides having an amino acid sequence at least 80% identical, morepreferably at least 90% identical, and still more preferably 95%, 96%,97%, 98% or 99% identical to those described in (a), (b), (c) or (d)above, as well as polypeptides having an amino acid sequence with atleast 90% similarity, and more preferably at least 95% similarity, tothose above are also provided.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which comprises the amino acid sequence of anepitope-bearing portion of a CKα-5 polypeptide having an amino acidsequence described in (a), (b), (c) or (d), above. Peptides orpolypeptides having the amino acid sequence of an epitope-bearingportion of a CKα-5 polypeptide of the invention include portions of suchpolypeptides with at least six (6) or seven (7), preferably at leastnine (9), and more preferably at least about 30 amino acids to about 50amino acids, although epitope-bearing polypeptides of any length up toand including the entire amino acid sequence of a polypeptide of theinvention described above also are included in the invention.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to a CKα-5 polypeptide having an amino acid sequencedescribed in (a), (b), (c) or (d) above. The invention further providesmethods for isolating antibodies that bind specifically to a CKα-5polypeptide having an amino acid sequence as described herein. Suchantibodies are useful diagnostically or therapeutically as describedbelow.

The invention also provides for pharmaceutical compositions comprisingCKα-5 polypeptides, particularly human CKα-5 polypeptides, which may beemployed, for instance, to stimulate wound healing, to treat solidtumors, microbial infections, autoimmune diseases, liver cirrhosis,osteoarthritis and pulmonary fibrosis. Methods of treating individualsin need of CKα-5 polypeptides are also provided.

The invention further provides compositions comprising a CKα-5polynucleotide or a CKα-5 polypeptide for administration to cells invitro, to cells ex vivo and to cells in vivo, or to a multicellularorganism. In certain particularly preferred embodiments of this aspectof the invention, the compositions comprise a CKα-5 polynucleotide forexpression of a CKα-5 polypeptide in a host organism for treatment ofdisease. Particularly preferred in this regard is expression in a humanpatient for treatment of a dysfunction associated with aberrantendogenous activity of a CKα-5.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a biological activity ofthe CKα-5 polypeptide, which involves contacting a receptor which isenhanced by the CKα-5 polypeptide with the candidate compound in thepresence of a CKα-5 polypeptide, assaying calcium mobilization orchemotactic activity of the cell expressing the receptor in the presenceof the candidate compound and the CKα-5 polypeptide, and comparing thereceptor activity to a standard level of activity, the standard beingassayed when contact is made between the receptor and the CKα-5polypeptide in the absence of the candidate compound. In this assay, anincrease in calcium mobilization or chemotaxis over the standardindicates that the candidate compound is an agonist of CKα-5 activityand a decrease in calcium mobilization or chemotaxis compared to thestandard indicates that the compound is an antagonist of CKα-5 activity.

It has been discovered that CKα-5 is expressed not only in neutrophilsbut also in monocytes, macrophages, liver, lung, testes, cerebellum andpineal gland. Therefore, nucleic acids of the invention are useful ashybridization probes for differential identification of the tissue(s) orcell type(s) present in a biological sample. Similarly, polypeptides andantibodies directed to those polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). In addition, for a number of disorders of the abovetissues or cells, particularly of the immune system, significantlyhigher or lower levels of CKα-5 gene expression may be detected incertain tissues (e.g., cancerous and wounded tissues) or bodily fluids(e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken froman individual having such a disorder, relative to a “standard” CKα-5gene expression level, i.e., the CKα-5 expression level in healthytissue from an individual not having the immune system disorder. Thus,the invention provides a diagnostic method useful during diagnosis ofsuch a disorder, which involves: (a) assaying CKα-5 gene expressionlevel in cells or body fluid of an individual; (b) comparing the CKα-5gene expression level with a standard CKα-5 gene expression level,whereby an increase or decrease in the assayed CKα-5 gene expressionlevel compared to the standard expression level is indicative ofdisorder in the immune system.

An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of CKα-5 activityin the body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an isolated CKα-5polypeptide of the invention or an agonist thereof.

A still further aspect of the invention is related to a method fortreating an individual in need of a decreased level of CKα-5 activity inthe body comprising, administering to such an individual a compositioncomprising a therapeutically effective amount of a CKα-5 antagonist.Preferred antagonists for use in the present invention areCKα-5-specific antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A–C shows the nucleotide sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:2) of CKα-5. The predicted leader sequence ofabout 27 amino acids is underlined. Note that the methionine residue atthe beginning of the leader sequence in FIG. 1 is shown in positionnumber (positive) 1, the leader positions in the corresponding sequenceof SEQ ID NO:2 are designated with positive position numbers. Thus, theleader sequence positions 1 to 27 in FIG. 1 correspond to positions—1 to27 in SEQ ID NO:2.

FIG. 2 shows the regions of identity between the amino acid sequence ofthe CKα-5 protein and translation product of the rat mRNA for MIP1-β(SEQ ID NO:3), as determined by Bestfit (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711) using the defaultparameters.

FIG. 3 shows an analysis of the CKα-5 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown. In the “Antigenic Index—Jameson-Wolf” graph, the positive peaksindicate locations of the highly antigenic regions of the CKα-5 protein,i.e., regions from which epitope-bearing peptides of the invention canbe obtained.

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a CKα-5 polypeptide having theamino acid sequence shown in SEQ ID NO:2, which was determined bysequencing cloned cDNAs. The nucleotide sequence shown in FIG. 1 (SEQ IDNO:1) was obtained by sequencing two cDNA clones: HMSCJ62 and HNFEM05.HMSCJ62 contains the full-length sequence shown in FIG. 1 and isdeposited at Human Genome Sciences, Inc., 9410 Key West Ave., Rockville,Md. 20850. HNFEM05 contains all but the first 360 nucleotides (includingall of the coding region) and was deposited as American Type CultureCollection (“ATCC®”) Deposit No. 209231 on Aug. 29, 1997. The ATCC® islocated at 10801 University Boulevard, Manassas, Va. 20110-2209.

The deposited clones are contained in the pBluescript SK(−) plasmid(Stratagene, La Jolla, Calif.).

The polypeptide of the present invention has amino acid sequencehomology to known chemokines, including the conserved C—X—C cysteinepattern characteristic of the alpha subfamily of chemokines beginningwith the first cysteine from the amino terminus in SEQ ID NO:2. TheCKα-5 protein of the present invention also shares sequence homologywith other chemokines including in particular the translation product ofthe human mRNA for MIP1-β (SEQ ID NO:3) as shown in FIG. 2.

Of the known members of the alpha chemokine family, the majority containan ELR motif (e.g., IL-8, ENA-78, GCP2, GRO-α, PBP, CTAP-III and NAP-2)and others lack the ELR motif (IP-10, PF4 and MIG). CKα-5 lacks the ELRmotif immediately preceding the first cysteine residue. It has beenclearly shown that this ELR motif is required for the neutrophil andendothelial cell chemotactic activity as well as the angiogenic activityof IL-8 (Strieter et al., J. Biol. Chem., 270:27348–27357 (1995)). Inaddition, it has been shown that the ELR-C—X—C chemokines have in vitroand in vivo angiogenic activity, whereas the C—X—C chemokines lackingthe ELR motif are actually angiostatic (Strieter et al., supra;Angiolillo et al., J. Exp. Med., 182:155–162 (1995); and Koch et al.,Science, 258:1798–1801 (1992)). In terms of a possible role of suchfactors in tumor angiogenesis, Smith et al., J. Exp. Med., 179:1409–1415(1994), has reported increased IL-8 levels in bronchogeneic carcinomatumor tissues which appear to be produced from the tumor cells.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc., FosterCity, Calif.), and all amino acid sequences of polypeptides encoded byDNA molecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

By “nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U), where eachthymidine deoxyribonucleotide (T) in the specified deoxyribonucleotidesequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequencein FIG. 1 (SEQ ID NO:1), a nucleic acid molecule of the presentinvention encoding a CKα-5 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative of the invention, the nucleicacid molecule described in FIG. 1 (SEQ ID NO:1) was discovered in cDNAlibraries derived primarily from immune-system tissues. Clone HMSCJ62was isolated from a monocyte cDNA library and HNFEM05 was isolated froma neutrophil cDNA library.

Additional clones of the same gene were also identified in cDNAlibraries from the following human tissues: colon and osteoclastoma.

The determined nucleotide sequence of the CKα-5 cDNA of FIG. 1 (SEQ IDNO:1) contains an open reading frame encoding a protein of 254 aminoacid residues, with an initiation codon at nucleotide positions 542–544of the nucleotide sequence in FIG. 1 (SEQ ID NO:1). The amino acidsequence of the CKα-5 protein shown in SEQ ID NO:2 is about 51% similarand 28% identical to the rat Macrophage Inflammatory Protein-1 β (FIG.2), which can be accessed on GenBank as Accession No. gi|459148.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, the actual complete CKα-5 polypeptideencoded by the deposited cDNA, which comprises about 254 amino acids,may be somewhat longer or shorter. More generally, the actual openreading frame may be anywhere in the range of ±20 amino acids, morelikely in the range of ±10 amino acids, of that predicted from themethionine codon at the N-terminus shown in FIG. 1 (SEQ ID NO:1).

Leader and Mature Sequences

The amino acid sequence of the complete CKα-5 protein includes a leadersequence and a mature protein, as shown in SEQ ID NO:2. More inparticular, the present invention provides nucleic acid moleculesencoding a mature form of the CKα-5 protein. Thus, according to thesignal hypothesis, once export of the growing protein chain across therough endoplasmic reticulum has been initiated, proteins secreted bymammalian cells have a signal or secretory leader sequence which iscleaved from the complete polypeptide to produce a secreted “mature”form of the protein. Most mammalian cells and even insect cells cleavesecreted proteins with the same specificity. However, in some cases,cleavage of a secreted protein is not entirely uniform, which results intwo or more mature species of the protein. Further, it has long beenknown that the cleavage specificity of a secreted protein is ultimatelydetermined by the primary structure of the complete protein, that is, itis inherent in the amino acid sequence of the polypeptide. Therefore,the present invention provides a nucleotide sequence encoding the matureCKα-5 polypeptide having the amino acid sequence encoded by the cDNAclone identified as ATCC® Deposit No. 209231. By the “mature CKα-5polypeptide having the amino acid sequence encoded by the cDNA clone inATCC® Deposit No. 209231” is meant the mature form(s) of the CKα-5protein produced by expression in a mammalian cell (e.g., COS cells, asdescribed below) of the complete open reading frame encoded by the humanDNA sequence of the clone contained in the deposited vector.

In addition, methods for predicting whether a protein has a secretoryleader as well as the cleavage point for that leader sequence areavailable. For instance, the method of McGeoch (Virus Res. 3:271–286(1985)) uses the information from a short N-terminal charged region anda subsequent uncharged region of the complete (uncleaved) protein. Themethod of von Heinje (Nucleic Acids Res. 14:4683–4690 (1986)) uses theinformation from the residues surrounding the cleavage site, typicallyresidues −13 to +2 where +1 indicates the amino terminus of the matureprotein. The accuracy of predicting the cleavage points of knownmammalian secretory proteins for each of these methods is in the rangeof 75–80% (von Heinje, supra). However, the two methods do not alwaysproduce the same predicted cleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the completeCKα-5 polypeptide was analyzed by a computer program “PSORT”, availablefrom Dr. Kenta Nakai of the Institute for Chemical Research, KyotoUniversity (see K. Nakai and M. Kanehisa, Genomics 14:897–911 (1992)),which is an expert system for predicting the cellular location of aprotein based on the amino acid sequence. As part of this computationalprediction of localization, the methods of McGeoch and von Heinje areincorporated.” The computation analysis above predicted one potentialcleavage site within the complete amino acid sequence shown in SEQ IDNO:2; that is, between residues 27 and 28 in FIG. 1 and residues 27 and28 in SEQ ID NO:2.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically. By “isolated” the inventors intend tospecifically exclude very large pieces of genetic material (200 kb+)such as isolated chromosomes and yeast artificial chromosomes (YACs).

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 542–544 of the nucleotide sequence shown in FIG. 1(SEQ ID NO:1).

Also included are DNA molecules comprising the coding sequence for thepredicted mature CKα-5 protein shown at positions 28–254 of SEQ ID NO:2.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode a CKα-5 protein. Of course, the genetic code andspecies-specific codon preferences are well known in the art. Thus, itwould be routine for one skilled in the art to generate the degeneratevariants described above, for instance, to optimize codon expression fora particular host (e.g., change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

In another aspect, the invention provides isolated nucleic acidmolecules encoding the CKα-5 polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCC®Deposit No. 209231 on Aug. 29, 1997.

Preferably, this nucleic acid molecule will encode the maturepolypeptide encoded by the above-described deposited cDNA clone.

The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) or the nucleotidesequence of the CKα-5 cDNA contained in the above-described depositedclone, or a nucleic acid molecule having a sequence complementary to oneof the above sequences. Such isolated molecules, particularly DNAmolecules, are useful as probes for gene mapping, by in situhybridization with chromosomes, and for detecting expression of theCKα-5 gene in human tissue, for instance, by Northern blot analysis.

The present invention is further directed to nucleic acid moleculesencoding portions of the nucleotide sequences described herein as wellas to fragments of the isolated nucleic acid molecules described herein.In particular, the invention provides a polynucleotide comprising anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 1–1303, 542–1303, 622–1303, 625–1303, 628–1303,631–1303, 634–1303, 637–1303, 640–1303, 643–1303, 646–1303, 649–1303,652–1303, 652–787, 652–817, 652–847, 652–877, 652–907, 652–937, 652–967,652–997, 652–1027, 652–1057, 652–1087, 652–1117, 652–1147, 652–1177,652–1207, 652–1237, or 652–1267 of SEQ ID NO:1.

In addition, the following nucleic acid molecules having nucleotidesequences related to extensive portions of SEQ ID NO:1 have beenidentified: HNFCO09R (SEQ ID NO:4) from clone HNFCO09; HNEDS26R (SEQ IDNO:5) from clone HNEDS26R; HOABD47R (SEQ ID NO:6) from clone HOABD47;HTEIR41R (SEQ ID NO:7) from clone HTEIR41; HMNAD36R (SEQ ID NO:8) fromclone HMNAD36; and HTEDQ07RA (SEQ ID NO:20) from clone HTEDQ07.

The following publicly available expressed sequence tags (“ESTs”), whichrelate to portions of SEQ ID NO:1, have also been identified: GenBankAccession No. AA130776 (SEQ ID NO:9); GenBank Accession No. AA290712(SEQ ID NO:10); GenBank Accession No. AA366329 (SEQ ID NO:11); GenBankAccession No. AA121716 (SEQ ID NO:12); GenBank Accession No. AA146672(SEQ ID NO:13); GenBank Accession No. AA149359 (SEQ ID NO:14); GenBankAccession No. AA569970 (SEQ ID NO:21); and GenBank Accession No.AA994552 (SEQ ID NO:22).

Polynucleotides of the invention preferably are not, and do notcomprise, one or more of the forgoing sequences 1–14 and 20–22.

Further, the invention includes a polynucleotide comprising any portionof at least about 17 nucleotides, preferably at least about 20, 25, or30, and most preferably at least about 50 nucleotides, of SEQ ID NO:1from nucleotide 542 to 1303.

The invention also includes a polynucleotide comprising any portion ofat least about 500, preferably about 600 nucleotides, of SEQ ID NO:1,preferably from residue 542 to 1,303. Particularly preferred nucleotidefragments in this regard include the fragment from nucleotide 622 to1,303 in SEQ ID NO:1.

More generally, by a fragment of an isolated nucleic acid moleculehaving the nucleotide sequence of the deposited cDNA or the nucleotidesequence shown in FIG. 1 (SEQ ID NO:1) is intended fragments at leastabout 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50–1,000 nt in lengthare also useful according to the present invention as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in FIG. 1 (SEQ ID NO:1). By a fragment atleast 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from the nucleotide sequence of thedeposited cDNA or the nucleotide sequence as shown in FIG. 1 (SEQ IDNO:1). Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the CKα-5polypeptide as identified from the Jameson-Wolf antigenic index shown inFIG. 3 and described in more detail below.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC® Deposit No. 209231. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30–70 (e.g., 50) nt of the reference polynucleotide. These areuseful as diagnostic probes and primers as discussed above and in moredetail below.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIG. 1 (SEQ ID NO:1)). Ofcourse, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the CKα-5 cDNA shown in FIG. 1(SEQ ID NO:1)), or to a complementary stretch of T (or U) residues,would not be included in a polynucleotide of the invention used tohybridize to a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode a CKα-5 polypeptide may include, but are not limited to thoseencoding the amino acid sequence of the mature polypeptide, by itself;and the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 27 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro- protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences.

Also encoded by nucleic acids of the invention are the above proteinsequences together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; anadditional coding sequence which codes for additional amino acids, suchas those which provide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821–824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the CKα-5 fusedto Fc at the N- or C-terminus.

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the CKα-5 protein. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the CKα-5 protein or portions thereof. Alsoespecially preferred in this regard are conservative substitutions.

Most highly preferred are nucleic acid molecules encoding the matureprotein having the amino acid sequence shown in SEQ ID NO:2 or themature CKα-5 amino acid sequence encoded by the deposited cDNA cloneeither of which may be modified so as to encode an amino terminalmethionine or other N- or C-terminal fusion peptides or polypeptides.

Further embodiments include an isolated polynucleotide comprising anucleotide sequence at least 90% identical, and more preferably at least95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selectedfrom the group consisting of: (a) a nucleotide sequence encoding theCKα-5 polypeptide shown as SEQ ID NO:2; (b) a nucleotide sequenceencoding the CKα-5 polypeptide shown as residues 1–254 of SEQ ID NO:2;(c) a nucleotide sequence encoding the predicted mature CKα-5 shown asresidues 28–254 in SEQ ID NO:2; (d) a nucleotide sequence encoding thepredicted extracellular domain of the CKα-5 polypeptide shown asresidues 28–205 in SEQ ID NO:2; (e) a nucleotide sequence encoding theCKα-5 polypeptide having the complete amino acid sequence encoded by thecDNA clone contained in ATCC® Deposit No. 209231; (t) a nucleotidesequence encoding the CKα-5 polypeptide having the complete amino acidsequence excepting the N terminal methionine encoded by the cDNA clonecontained in ATCC® Deposit No. 209231; (g) a nucleotide sequenceencoding the mature CKα-5 polypeptide having the amino acid sequenceencoded by the cDNA clone contained in ATCC® Deposit No. 209231; (h) anucleotide sequence encoding the extracellular domain of the CKα-5polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC® Deposit No. 209231; and (i) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g) or (h) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h) or (i) above, or a polynucleotide which hybridizesunder stringent hybridization conditions to a polynucleotide in (a),(b), (c), (d), (e), (f), (g), (h) or (i), above. This polynucleotidewhich hybridizes does not hybridize under stringent hybridizationconditions to a polynucleotide having a nucleotide sequence consistingof only A residues or of only T residues. An additional nucleic acidembodiment of the invention relates to an isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a CKα-5 polypeptide having an amino acidsequence in (a), (b), (c), (d), (e), (f), (g) or (h) above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofCKα-5 polypeptides or peptides by recombinant techniques.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a CKα-5polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the CKα-5polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIG. 1 or to the nucleotides sequence ofthe deposited cDNA clone can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482–489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid sequence of thedeposited cDNA, irrespective of whether they encode a polypeptide havingCKα-5 activity. This is because even where a particular nucleic acidmolecule does not encode a polypeptide having CKα-5 activity, one ofskill in the art would still know how to use the nucleic acid molecule,for instance, as a hybridization probe or a polymerase chain reaction(PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having CKα-5 activityinclude, inter alia, (1) isolating the CKα-5 gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe CKα-5 gene, as described in Verma et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York (1988); andNorthern Blot analysis for detecting CKα-5 mRNA expression in specifictissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide having CKα-5protein activity. By “a polypeptide having CKα-5 activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the extracellular portion of the complete CKα-5protein. One such activity believed to be possessed by such polypeptidesis the ability to modulate colony formation of bone marrow progenitorcells, as does Human Chemokine HCC-1. An in vitro colony forming assayfor measuring the extent of inhibition of myeloid progenitor cells isdescribed in Young et al., The Journal of Immunology 155:2661–2667(1995). Briefly, the assay involves collecting human or mouse bonemarrow cells and plating the same on agar, adding one or more growthfactors and either (1) transfected host cell-supernatant containingCKα-5 protein (or a candidate polypeptide) or (2) nontransfected hostcell-supernatant control, and measuring the effect on colony formationby murine and human CFU-granulocyte-macrophages (CFU-GM), by humanburst-forming unit-erythroid (BFU-E), or by human CFUgranulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM). Such activityis useful for protecting myeloid progenitor cells during chemotherapy.

CKα-5 protein modulates immune system cell proliferation anddifferentiation in a dose-dependent manner in the above-described assay.Thus, “a polypeptide having CKα-5 protein activity” includespolypeptides that also exhibit any of the same myeloid progenitormodulation activities in the above-described assays in a dose-dependentmanner. Although the degree of dose-dependent activity need not beidentical to that of the CKα-5 protein, preferably, “a polypeptidehaving CKα-5 protein activity” will exhibit substantially similardose-dependence in a given activity as compared to the CKα-5 protein(i.e., the candidate polypeptide will exhibit greater activity or notmore than about 25-fold less and, preferably, not more than abouttenfold less activity relative to the reference CKα-5 protein).

Like other CXC chemokines, CKα-5 exhibits activity on leukocytesincluding for example monocytes, lymphocytes and neutrophils. For thisreason CKα-5 is active in directing the proliferation, differentiationand migration of these cell types. Such activity is useful for immuneenhancement or suppression, myeloprotection, stem cell mobilization,acute and chronic inflammatory control and treatment of leukemia. Assaysfor measuring such activity are known in the art. For example, seePeters et al., Immun. Today 17:273 (1996); Young et al., J. Exp. Med.182:1111 (1995); Caux et al., Nature 390:258 (1992); andSantiago-Schwarz et al., Adv. Exp. Med. Biol. 378:7 (1995).” The assaysand protocols from each of the forgoing journal articles areincorporated herein by reference.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1) will encode apolypeptide “having CKα-5 protein activity.” In fact, since degeneratevariants of these nucleotide sequences all encode the same polypeptide,this will be clear to the skilled artisan even without performing theabove described comparison assay. It will be further recognized in theart that, for such nucleic acid molecules that are not degeneratevariants, a reasonable number will also encode a polypeptide havingCKα-5 protein activity. This is because the skilled artisan is fullyaware of amino acid substitutions that are either less likely or notlikely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid), as furtherdescribed below.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of CKα-5polypeptides or fragments thereof by recombinant techniques. The vectormay be, for example, a phage, plasmid, viral or retroviral vector.Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe transcripts expressed by the constructs will preferably include atranslation initiating codon at the beginning and a termination codon(UAA, UGA or UAG) appropriately positioned at the end of the polypeptideto be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc., supra; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. Other suitable vectors will bereadily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., J. Molecular Recognition 8:52–58 (1995) and K.Johanson et al., J. Biol. Chem. 270:9459–9471 (1995).

The CKα-5 protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention include: products purified from natural sources, includingbodily fluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

Polypeptides and Fragments

The invention further provides an isolated CKα-5 polypeptide having theamino acid sequence encoded by the deposited cDNA, or the amino acidsequence in SEQ ID NO:2, or a peptide or polypeptide comprising aportion of the above polypeptides.

Variant and Mutant Polypeptides

To improve or alter the characteristics of CKα-5 polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

N-Terminal and C-Terminal Deletion Mutants

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron et al., J. Biol. Chem., 268:2984–2988 (1993)reported modified KGF proteins that had heparin binding activity even if3, 8, or 27 amino-terminal amino acid residues were missing. In thepresent case, since the protein of the invention is a member of thechemokine polypeptide family, deletions of N-terminal amino acids up tothe first “Cys” required for formation of a disulfide bridge may retainsome biological activity such as receptor binding or modulation oftarget cell activities. Polypeptides having further N-terminal deletionsincluding the cysteine residue at position 38 in FIG. 1 (position 11 inSEQ ID NO:2) would not be expected to retain such biological activitiesbecause it is known that this residue in a chemokine-related polypeptideis required for forming a disulfide bridge to provide structuralstability which is needed for receptor binding and signal transduction.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature form of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the CKα-5 protein shown in SEQ ID NO:2, up to the cysteineresidue at position number 38, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues n-205 of SEQ ID NO:2,where n is an integer in the range of 24–38, and Cys-38 is the positionof the first residue from the N-terminus of the extracellular domain ofthe CKα-5 polypeptide (shown in SEQ ID NO:2) believed to be required forreceptor binding activity of the CKα-5 protein.

More in particular, the invention provides polypeptides comprising anamino acid sequence selected from the group consisting of: 24 to 205, 25to 205, 26 to 205, 27 to 205, 28 to 205, 29 to 205, 30 to 205, 31 to205, 32 to 205, 33 to 205, 34 to 205, 35 to 205, 36 to 205, 37 to 205,and 38 to 205, all of SEQ ID NO:2. Polynucleotides encoding suchpolypeptides are also provided.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, Interferon gamma shows up to ten timeshigher activities by deleting 8–10 amino acid residues from the carboxyterminus of the protein (Döbeli et al., J. Biotechnology 7:199–216(1988). In the present case, since the protein of the invention is amember of the chemokine polypeptide family, deletions of C-terminalamino acids up to the cysteine at position 82 of SEQ ID NO:2 may retainsome biological activity such as receptor binding or modulation oftarget cell activities. Polypeptides having further C-terminal deletionsincluding the cysteine residue at position 82 of FIG. 1 (82 of SEQ IDNO:2) would not be expected to retain such biological activities becauseit is known that this residue in a chemokine-related polypeptide isrequired for forming a disulfide bridge to provide structural stabilitywhich is needed for receptor binding and signal transduction.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature form of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Accordingly, the present invention further provides polypeptides havingone or more residues from the carboxy terminus of the amino acidsequence of the CKα-5 shown in SEQ ID NO:2, up to the cysteine residueat position 82 of SEQ ID NO:2, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptideshaving the amino acid sequence of residues (24)-m of the amino acidsequence in SEQ ID NO:2, where m is any integer in the range of 82 to205, and residue cysteine-82 is the position of the first residue fromthe C-terminus of the complete CKα-5 polypeptide (shown in SEQ ID NO:2)believed to be required for receptor binding and target cell modulationactivities of the CKα-5 protein.

More in particular, the invention provides polypeptides comprising anamino acid sequence selected from the group consisting of: 24 to 82, 24to 83, 24 to 84, 24 to 85, 24 to 86, 24 to 87, 24 to 88, 24 to 89, 24 to90, 24 to 91, 24 to 92, 24 to 93, 24 to 94, 24 to 95, 24 to 96, 24 to97, 24 to 98, 24 to 99, 24 to 100, 24 to 101, 24 to 102, 24 to 103, 24to 104, 24 to 105, 24 to 106, 24 to 107, 24 to 108, 24 to 109, 24 to110, 24 to 111, 24 to 112, 24 to 113, 24 to 114, 24 to 115, 24 to 116,24 to 117, 24 to 118, 24 to 119, 24 to 120, 24 to 121, 24 to 122, 24 to123, 24 to 124, 24 to 125, 24 to 126, 24 to 127, 24 to 128, 24 to 129,24 to 130, 24 to 131, 24 to 132, 24 to 133, 24 to 134, 24 to 135, 24 to136, 24 to 137, 24 to 138, 24 to 139, 24 to 140, 24 to 141, 24 to 142,24 to 143, 24 to 144, 24 to 145, 24 to 146, 24 to 147, 24 to 148, 24 to149, 24 to 150, 24 to 151, 24 to 152, 24 to 153, 24 to 154, 24 to 155,24 to 156, 24 to 157, 24 to 158, 24 to 159, 24 to 160, 24 to 161, 24 to162, 24 to 163, 24 to 164, 24 to 165, 24 to 166, 24 to 167, 24 to 168,24 to 169, 24 to 170, 24 to 171, 24 to 172, 24 to 173, 24 to 174, 24 to175, 24 to 176, 24 to 177, 24 to 178, 24 to 179, 24 to 180, 24 to 181,24 to 182, 24 to 183, 24 to 184, 24 to 185, 24 to 186, 24 to 187, 24 to188, 24 to 189, 24 to 190, 24 to 191, 24 to 192, 24 to 193, 24 to 194,24 to 195, 24 to 196, 24 to 196, 24 to 197, 24 to 198, 24 to 199, 24 to200, 24 to 201, 24 to 202, 24 to 203, 24 to 204, and 24 to 205, all ofSEQ ID NO:2. Polynucleotides encoding these polypeptides also areprovided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of the soluble form(extracellular domain), which may be described generally as havingresidues n-m of SEQ ID NO:2, where n and m are integers as describedabove. Particularly preferred are polypeptides comprising an amino acidsequence selected from the group consisting of: 38 to 82, 38 to 83, 37to 84, 37 to 85, 37 to 86, 37 to 87, 37 to 88, 37 to 89, 36 to 90, 36 to91, 36 to 92, 36 to 93, 36 to 94, 36 to 95, 36 to 96, 36 to 97, 36 to98, 36 to 99, 36 to 100, 35 to 101, 35 to 102, 35 to 103, 35 to 104, 35to 105, 35 to 106, 35 to 107, 35 to 108, 35 to 109, 35 to 110, 35 to111, 34 to 112, 34 to 113, 34 to 114, 34 to 115, 34 to 116, 34 to 117,34 to 118, 34 to 119, 34 to 120, 34 to 121, 34 to 122, 34 to 123, 34 to124, 34 to 125, 34 to 126, 34 to 127, 33 to 128, 33 to 129, 33 to 130,33 to 131, 33 to 132, 33 to 133, 33 to 134, 33 to 135, 33 to 136, 33 to137, 33 to 138, 33 to 139, 33 to 140, 33 to 141, 33 to 142, 33 to 143,33 to 144, 33 to 145, 33 to 146, 33 to 147, 33 to 148, 33 to 149, 33 to150, 33 to 151, 33 to 152, 33 to 153, 33 to 154, 33 to 155, 33 to 156,33 to 157, 33 to 158, 33 to 159, 33 to 160, 33 to 161, 33 to 162, 33 to163, 33 to 164, 33 to 165, 33 to 166, 33 to 167, 33 to 168, 33 to 169,33 to 170, 33 to 171, 33 to 172, 33 to 173, 33 to 174, 33 to 175, 33 to176, 33 to 177, 33 to 178, 33 to 179, 33 to 180, 33 to 181, 33 to 182,33 to 183, 33 to 184, 32 to 185, 32 to 186, 32 to 187, 32 to 188, 32 to189, 32 to 190, 32 to 191, 32 to 192, 32 to 193, 32 to 194, 32 to 195,32 to 196, 31 to 197, 31 to 198, 31 to 199, 31 to 200, 31 to 201, 31 to202, 31 to 203, 31 to 204, and 31 to 205, all of SEQ ID NO:2.Polynucleotides encoding these polypeptides also are provided.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete CKα-5 amino acid sequenceencoded by the cDNA clone contained in ATCC® Deposit No. 209231, wherethis portion excludes from 23 to about 37 amino acids from the aminoterminus of the extracellular domain of the CKα-5 polypeptide encoded bythe cDNA clone contained in ATCC® Deposit No. 209231, or from 49 toabout 172 amino acids from the carboxy terminus, or any combination ofthe above amino terminal and carboxy terminal deletions, of theextracellular domain encoded by the cDNA clone contained in ATCC®Deposit No. 209231. Polynucleotides encoding all of the above deletionmutant polypeptide forms also are provided.

Other Mutants

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the CKα-5 polypeptide can be varied withoutsignificant effect of the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the CKα-5 polypeptidewhich show substantial CKα-5 polypeptide activity or which includeregions of CKα-5 protein such as the protein portions discussed below.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions selected according to general rules known in the artso as have little effect on activity. For example, guidance concerninghow to make phenotypically silent amino acid substitutions is providedin Bowie, J. U. et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306–1310 (1990),wherein the authors indicate that there are two main approaches forstudying the tolerance of an amino acid sequence to change. The firstmethod relies on the process of evolution, in which mutations are eitheraccepted or rejected by natural selection. The second approach usesgenetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections or screens to identifysequences that maintain functionality.

As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, J. U. et al., supra, and thereferences cited therein. Typically seen as conservative substitutionsare those shown in Table 1.

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the above form of the polypeptide, such as an IgG Fc fusionregion peptide or leader or secretory sequence or a sequence which isemployed for purification of the above form of the polypeptide or aproprotein sequence. Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein.

Thus, the CKα-5 of the present invention may include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein (seeTable 1).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the CKα-5 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081–1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro or in vitro proliferativeactivity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331–340 (1967);Robbins et al., Diabetes 36: 838–845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307–377 (1993).

Replacement of amino acids can also change the selectivity of thebinding of a ligand to cell surface receptors. For example, Ostade etal., Nature 361:266–268 (1993) describes certain mutations resulting inselective binding of TNF-α to only one of the two known types of TNFreceptors. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899–904 (1992) and de Vos et al. Science 255:306–312 (1992)).

As described above CKα-5 lacks an ELR motif. By making a specificmutation in CKα-5 to include an ELR motif in the position where such amotif is typically found in related chemokines, CKα-5 will act as anantagonist, thus possessing angiogenic activity. Accordingly,polypeptides of the present invention include CKα-5-ELR mutants. SuchCKα-5-ELR mutants are comprised of the full-length or preferably themature CKα-5 protein. Further, since CKα-5 is a member of thechemokine-related protein family, to modulate rather than completelyeliminate biological activities of CKα-5 preferably mutations are madein sequences encoding amino acids in the CKα-5 extracellular domain,i.e., in positions 28 to 205 of SEQ ID NO:2, more preferably in residueswithin this region which are not conserved in all members of thechemokine family. As is known in the art, the four spatially conservedcysteines present in all chemokines, positions 38, 40, 68 and 82 inCKα-5, are required for the formation of two disulfide bridges. Thus, itis preferable not to alter any of the four cysteine residues located atpositions 38, 40, 68 and 82 in SEQ ID NO:2. Also forming part of thepresent invention are isolated polynucleotides comprising nucleic acidsequences which encode the above CKα-5 mutants.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the CKα-5 polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31–40 (1988). Polypeptides of the invention also can bepurified from natural or recombinant sources using anti-CKα-5 antibodiesof the invention in methods which are well known in the art of proteinpurification.

The invention further provides an isolated CKα-5 polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of the full-length CKα-5 polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:2 or the complete aminoacid sequence encoded by the cDNA clone contained in the ATCC® DepositNo. 209231; (b) the amino acid sequence of the full-length CKα-5polypeptide having the complete amino acid sequence shown in SEQ ID NO:2excepting the N-terminal methionine (i.e., positions 2 to 254 of SEQ IDNO:2) or the complete amino acid sequence excepting the N terminalmethionine encoded by the cDNA clone contained in the ATCC® Deposit No.209231; (c) the amino acid sequence of the mature CKα-5 polypeptidehaving the amino acid sequence shown in SEQ ID NO:2 as residues 28–254or the amino acid sequence of the mature CKα-5 encoded by the cDNA clonecontained in the ATCC® Deposit No. 209231; and (d) the amino acidsequence of the extracellular domain of the CKα-5 polypeptide having theamino acid sequence shown in SEQ ID NO:2 as residues 28–205 or the aminoacid sequence of the extracellular domain of the CKα-5 polypeptideencoded by the cDNA clone contained in the ATCC® Deposit No. 209231.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above. The polypeptides of the invention also comprisethose which are at least 80% identical, more preferably at least 90% or95% identical, still more preferably at least 96%, 97%, 98% or 99%identical to the polypeptide encoded by the deposited cDNA or to thepolypeptide of SEQ ID NO:2, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids. Polynucleotides encoding such variant polypeptides arealso provided.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482–489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a CKα-5polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the CKα-5 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or to the amino acid sequence encodedby deposited cDNA clone can be determined conventionally using knowncomputer programs such the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference amino acid sequence and that gaps in homology ofup to 5% of the total number of amino acid residues in the referencesequence are allowed.

The polypeptide of the present invention could be used as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

As described in detail below, the polypeptides of the present inventioncan also be used to raise polyclonal and monoclonal antibodies, whichare useful in assays for detecting CKα-5 protein expression as describedbelow or as agonists and antagonists capable of enhancing or inhibitingCKα-5 protein function. Further, such polypeptides can be used in theyeast two-hybrid system to “capture” CKα-5 protein binding proteinswhich are also candidate agonists and antagonists according to thepresent invention. The yeast two hybrid system is described in Fieldsand Song, Nature 340:245–246 (1989).

Epitope-Bearing Portions

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998–4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) “Antibodies that react withpredetermined sites on proteins,” Science, 219:660–666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals. Antigenic epitope-bearing peptides and polypeptidesof the invention are therefore useful to raise antibodies, includingmonoclonal antibodies, that bind specifically to a polypeptide of theinvention. See, for instance, Wilson et al., Cell 37:767–778 (1984) at777.

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples of antigenic polypeptides or peptidesthat can be used to generate CKα-5-specific antibodies include: apolypeptide comprising amino acid residues from about Tyr-39 to aboutSer-51 in SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Cys-82 to about Val-90 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Thr-91 to about Pro-110 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about Ser-120 to aboutIle-145; a polypeptide comprising amino acid residues from about Asp-4to about Ser-9; a polypeptide comprising amino acid residues from aboutLys-42 to about Pro-50; a polypeptide comprising amino acid residuesfrom about Gly-83 to about Pro-88; a polypeptide comprising amino acidresidues from about Glu-128 to about Asp-133; a polypeptide comprisingamino acid residues from about Ser-159 to about Thr-170; a polypeptidecomprising amino acid residues from about Glut-184 to about Pro-198; apolypeptide comprising amino acid residues from about Lys-228 to aboutAsp-240; and a polypeptide comprising amino acid residues from aboutPro-250 to about Asn-253. These polypeptide fragments have beendetermined to bear antigenic epitopes of the CKα-5 protein by theanalysis of the Jameson-Wolf antigenic index, as shown in FIG. 3, above.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. See, e.g., Houghten, R. A. (1985)“General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids.” Proc. Natl. Acad. Sci. USA 82:5131–5135; this“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910–914; and Bittle, F. J. et al., J.Gen. Virol. 66:2547–2354 (1985). Immunogenic epitope-bearing peptides ofthe invention, i.e., those parts of a protein that elicit an antibodyresponse when the whole protein is the immunogen, are identifiedaccording to methods known in the art. See, for instance, Geysen et al.,supra. Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describesa general method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimnotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C1–C7-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

Fusion Proteins

As one of skill in the art will appreciate, CKα-5 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EP A 394,827; Traunecker et al., Nature 331:84–86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric CKα-5 protein or proteinfragment alone (Fountoulakis et al., J. Biochem. 270:3958–3964 (1995)).

Antibodies

CKα-5-protein specific antibodies for use in the present invention canbe raised against the intact CKα-5 protein or an antigenic polypeptidefragment thereof, which may be presented together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse) or, if it is long enough (at least about 25 amino acids), withouta carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to CKα-5 protein. Fab and F(ab′)2 fragments lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J. Nucl. Med. 24:316–325 (1983)). Thus, thesefragments are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the CKα-5 protein oran antigenic fragment thereof can be administered to an animal in orderto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of CKα-5 protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or CKα-5 protein binding fragments thereof).Such monoclonal antibodies can be prepared using hybridoma technology(Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol.6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,(1981) pp. 563–681). In general, such procedures involve immunizing ananimal (preferably a mouse) with a CKα-5 protein antigen or, morepreferably, with a CKα-5 protein-expressing cell. Suitable cells can berecognized by their capacity to bind anti-CKα-5 protein antibody. Suchcells may be cultured in any suitable tissue culture medium; however, itis preferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP2O), available from the American TypeCulture Collection, Rockville, Md. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al. (Gastroenterology80:225–232 (1981)). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the CKα-5 protein antigen.

Alternatively, additional antibodies capable of binding to the CKα-5protein antigen may be produced in a two-step procedure through the useof anti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, CKα-5-protein specific antibodies are used to immunizean animal, preferably a mouse. The splenocytes of such an animal arethen used to produce hybridoma cells, and the hybridoma cells arescreened to identify clones which produce an antibody whose ability tobind to the CKα-5 protein-specific antibody can be blocked by the CKα-5protein antigen. Such antibodies comprise anti-idiotypic antibodies tothe CKα-5 protein-specific antibody and can be used to immunize ananimal to induce formation of further CKα-5 protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, CKα-5protein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of anti-CKα-5 in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).

Immune System-Related Disorders

Diagnosis

The present inventors have discovered that CKα-5 is expressed inneutrophils and monocytes. For a number of immune system-relateddisorders, substantially altered (increased or decreased) levels ofCKα-5 gene expression can be detected in immune system tissue or othercells or bodily fluids (e.g., sera, plasma, urine, synovial fluid orspinal fluid) taken from an individual having such a disorder, relativeto a “standard” CKα-5 gene expression level, that is, the CKα-5expression level in immune system tissues or bodily fluids from anindividual not having the immune system disorder. Thus, the inventionprovides a diagnostic method useful during diagnosis of an immune systemdisorder, which involves measuring the expression level of the geneencoding the CKα-5 protein in immune system tissue or other cells orbody fluid from an individual and comparing the measured gene expressionlevel with a standard CKα-5 gene expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of an immune system disorder.

In particular, it is believed that certain tissues in mammals withcancer, particularly osteosarcoma, express significantly altered levelsof the CKα-5 protein and mRNA encoding the CKα-5 protein when comparedto a corresponding “standard” level. Further, it is believed thataltered levels of the CKα-5 protein can be detected in certain bodyfluids (e.g., sera, plasma, urine, and spinal fluid) from mammals withsuch a cancer when compared to sera from mammals of the same species nothaving the cancer.

Thus, the invention provides a diagnostic method useful during diagnosisof an immune system disorder, including cancers of the skeletal andimmune systems, which involves measuring the expression level of thegene encoding the CKα-5 protein in immune or skeletal system tissue orother cells or body fluid from an individual and comparing the measuredgene expression level with a standard CKα-5 gene expression level,whereby an increase or decrease in the gene expression level compared tothe standard is indicative of an immune system disorder.

Where a diagnosis of a disorder in the immune system, includingdiagnosis of a tumor, has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced or depressed CKα-5 gene expressionwill experience a worse clinical outcome relative to patients expressingthe gene at a level nearer the standard level.

By “assaying the expression level of the gene encoding the CKα-5protein” is intended qualitatively or quantitatively measuring orestimating the level of the CKα-5 protein or the level of the mRNAencoding the CKα-5 protein in a first biological sample either directly(e.g., by determining or estimating absolute protein level or mRNAlevel) or relatively (e.g., by comparing to the CKα-5 protein level ormRNA level in a second biological sample). Preferably, the CKα-5 proteinlevel or mRNA level in the first biological sample is measured orestimated and compared to a standard CKα-5 protein level or mRNA level,the standard being taken from a second biological sample obtained froman individual not having the disorder or being determined by averaginglevels from a population of individuals not having a disorder of theimmune system. As will be appreciated in the art, once a standard CKα-5protein level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains CKα-5 protein or mRNA. As indicated, biological samplesinclude body fluids (such as sera, plasma, urine, synovial fluid andspinal fluid) which contain free CKα-5 protein, immune and skeletalsystem tissue, and other tissue sources found to express complete matureCKα-5 or a CKα-5 receptor. Methods for obtaining tissue biopsies andbody fluids from mammals are well known in the art. Where the biologicalsample is to include mRNA, a tissue biopsy is the preferred source.

The present invention is useful for diagnosis or treatment of variousimmune system-related disorders in mammals, preferably humans. Suchdisorders include tumors, cancers, sarcomas such as osteosarcoma,interstitial lung disease (such as Langerhans cell granulomatosis) andany disregulation of immune cell function including, but not limited to,autoimmunity, arthritis, leukemias, lymphomas, immunosuppression,immunity, humoral immunity, inflammatory bowel disease, myelosuppression, and the like.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156–159 (1987). Levels ofmRNA encoding the CKα-5 protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Assaying CKα-5 protein levels in a biological sample can occur usingantibody-based techniques. For example, CKα-5 protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976–985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087–3096 (1987)). Other antibody-basedmethods useful for detecting CKα-5 protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹²¹In), and technetium (^(99m)Tc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

In addition to assaying CKα-5 protein levels in a biological sampleobtained from an individual, CKα-5 protein can also be detected in vivoby imaging. Antibody labels or markers for in vivo imaging of CKα-5protein include those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may heincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A CKα-5 protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain CKα-5 protein. In vivotumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

Treatment

As noted above, CKα-5 polynucleotides and polypeptides are useful fordiagnosis of conditions involving abnormally high or low expression ofCKα-5 activities. Given the cells and tissues where CKα-5 is expressedas well as the activities modulated by CKα-5, it is readily apparentthat a substantially altered (increased or decreased) level ofexpression of CKα-5 in an individual compared to the standard or“normal” level produces pathological conditions related to the bodilysystem(s) in which CKα-5 is expressed and/or is active.

It will also be appreciated by one of ordinary skill that, since theCKα-5 protein of the invention is a member of the chemokine family themature secreted form of the protein may be released in soluble form fromthe cells which express the CKα-5 by proteolytic cleavage. Therefore,when CKα-5 is added from an exogenous source to cells, tissues or thebody of an individual, the protein will exert its physiologicalactivities on its target cells of that individual. Also, cellsexpressing this transmembrane protein may be added to cells, tissues orthe body of an individual and these added cells will bind to cellsexpressing receptor for CKα-5, whereby the cells expressing CKα-5 cancause actions, e.g., cell stimulation, on the receptor-bearing targetcells.

Therefore, it will be appreciated that conditions caused by a decreasein the standard or normal level of CKα-5 activity in an individual,particularly disorders of the immune system, can be treated byadministration of CKα-5 polypeptide in the form of the solubleextracellular domain or cells expressing the complete protein. Thus, theinvention also provides a method of treatment of an individual in needof an increased level of CKα-5 activity comprising administering to suchan individual a pharmaceutical composition comprising an amount of anisolated CKα-5 polypeptide of the invention, effective to increase theCKα-5 activity level in such an individual.

Since CKα-5 lacks an ELR motif, it also should be an angiostatic factorrather than an angiogenic factor. In addition, since CKα-5 inhibitsendothelial cell function, it will have a wide range ofanti-inflammatory activities. CKα-5 may be employed as ananti-neovascularizing agent to treat solid tumors by stimulating theinvasion and activation of host defense cells, e.g., cytotoxic T cellsand macrophages and by inhibiting the angiogeniesis of tumors. Those ofskill in the art will recognize other non-cancer indications where bloodvessel proliferation is not wanted. They may also be employed to enhancehost defenses against resistant chronic and acute infections, forexample, myobacterial infections via the attraction and activation ofmicrobicidal leukocytes. CKα-5 may also be employed to inhibit T-cellproliferation by the inhibition of IL-2 biosynthesis for the treatmentof T-cell mediated auto-immune diseases and lymphocytic leukemias. CKα-5may also be employed to stimulate wound healing, both via therecruitment of debris clearing and connective tissue promotinginflammatory cells and also via its control of excessive TGF -mediatedfibrosis. In this same manner, CKα-5 may also be employed to treat otherfibrotic disorders, including liver cirrhosis, osteoarthritis andpulmonary fibrosis. CKα-5 also increases the presence of eosinophilswhich have the distinctive function of killing the larvae of parasitesthat invade tissues, as in schistosomiasis, trichinosis and ascariasis.It may also be employed to regulate hematopoiesis, by regulating theactivation and differentiation of various hematopoietic progenitorcells, for example, to release mature leukocytes from the bone marrowfollowing chemotherapy, i.e., in stem cell mobilization CKα-5 may alsobe employed to treat sepsis. Also, by making a specific mutation toinclude an ELR motif, CKα-5-ELR will have angiogenic activities whichare useful for treating all of the disease states where angiogenesiswould be beneficial, i.e., to promote wound healing, re-vascularizationof damaged limbs from injury or disease, and others known to those ofskill in the art.

Formulations

The CKα-5 polypeptide composition will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with CKα-5 polypeptide alone), the site of deliveryof the CKα-5 polypeptide composition, the method of administration, thescheduling of administration, and other factors known to practitioners.The “effective amount” of CKα-5 polypeptide for purposes herein is thusdetermined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofCKα-5 polypeptide administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the CKα-5 polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1–4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

Pharmaceutical compositions containing the CKα-5 of the invention may beadministered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

The CKα-5 polypeptide is also suitably administered by sustained-releasesystems. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547–556(1983)), poly (2- hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167–277 (1981), and R. Langer, Chem. Tech.12:98–105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release CKα-5polypeptide compositions also include liposomally entrapped CKα-5polypeptide. Liposomes containing CKα-5 polypeptide are prepared bymethods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad.Sci. (USA) 82:3688–3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030–4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949;EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200–800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal CKα-5 polypeptide therapy.

For parenteral administration, in one embodiment, the CKα-5 polypeptideis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the CKα-5polypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The CKα-5 polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1–10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of CKα-5 polypeptide salts.

CKα-5 polypeptide to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic CKα-5polypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

CKα-5 polypeptide ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous CKα-5 polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized CKα-5 polypeptide using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the action of CKα-5 on cells, such as itsinteraction with CKα-5-binding molecules such as receptor molecules. Anagonist is a compound which increases the natural biological functionsof CKα-5 or which functions in a manner similar to CKα-5, whileantagonists decrease or eliminate such functions.

In another aspect of this embodiment the invention provides a method foridentifying a receptor protein or other ligand-binding protein whichbinds specifically to a CKα-5 polypeptide. For example, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds CKα-5. Thepreparation is incubated with labeled CKα-5 CKα-5 and complexes of CKα-5bound to the receptor or other binding protein are isolated andcharacterized according to routine methods known in the art.Alternatively, the CKα-5 polypeptide may be bound to a solid support sothat binding molecules solubilized from cells are bound to the columnand then eluted and characterized according to routine methods.

In the assay of the invention for agonists or antagonists, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds CKα-5, such asa molecule of a signaling or regulatory pathway modulated by CKα-5. Thepreparation is incubated with labeled CKα-5 in the absence or thepresence of a candidate molecule which may be a CKα-5 agonist orantagonist. The ability of the candidate molecule to bind the bindingmolecule is reflected in decreased binding of the labeled ligand.Molecules which bind gratuitously, i.e., without inducing the effects ofCKα-5 on binding the CKα-5 binding molecule, are most likely to be goodantagonists. Molecules that bind well and elicit effects that are thesame as or closely related to CKα-5 are agonists.

CKα-5-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that ofCKα-5 or molecules that elicit the same effects as CKα-5. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

Another example of an assay for CKα-5 antagonists is a competitive assaythat combines CKα-5 and a potential antagonist with membrane-bound CKα-5receptor molecules or recombinant CKα-5 receptor molecules underappropriate conditions for a competitive inhibition assay. CKα-5 can belabeled, such as by radioactivity, such that the number of CKα-5molecules bound to a receptor molecule can be determined accurately toassess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducingCKα-5-induced activities, thereby preventing the action of CKα-5 byexcluding CKα-5 from binding.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in Okano, J. Neurochem. 56: 560 (1991);“Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression.” CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA. For example, the 5′ coding portion of apolynucleotide that encodes the mature polypeptide of the presentinvention may be used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcriptionthereby preventing transcription and the production of CKα-5. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into CKα-5 polypeptide. Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of CKα-5 protein.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described above.

The antagonists may be employed for instance to inhibit the chemotaxisand activation of macrophages and their precursors, and of neutrophils,basophils, B lymphocytes and some T-cell subsets, e.g., activated andCD8 cytotoxic T cells and natural killer cells, in certain auto-immuneand chronic inflammatory and infective diseases. Examples of auto-immunediseases include multiple sclerosis, and insulin-dependent diabetes. Theantagonists may also be employed to treat infectious diseases includingsilicosis, sarcoidosis, idiopathic pulmonary fibrosis by preventing therecruitment and activation of mononuclear phagocytes. They may also beemployed to treat idiopathic hyper-eosinophilic syndrome by preventingeosinophil production and migration. Endotoxic shock may also be treatedby the antagonists by preventing the migration of macrophages and theirproduction of the human chemokine polypeptides of the present invention.The antagonists may also be employed for treating atherosclerosis, bypreventing monocyte infiltration in the artery wall. The antagonists mayalso be employed to treat histamine-mediated allergic reactions andimmunological disorders including late phase allergic reactions, chronicurticaria, and atopic dermatitis by inhibiting chemokine-induced mastcell and basophil degranulation and release of histamine. IgE-mediatedallergic reactions such as allergic asthma, rhinitis, and eczema mayalso be treated. The antagonists may also be employed to treat chronicand acute inflammation by preventing the attraction of monocytes to awound area. They may also be employed to regulate normal pulmonarymacrophage populations, since chronic and acute inflammatory pulmonarydiseases are associated with sequestration of mononuclear phagocytes inthe lung. Antagonists may also be employed to treat rheumatoid arthritisby preventing the attraction of monocytes into synovial fluid in thejoints of patients. Monocyte influx and activation plays a significantrole in the pathogenesis of both degenerative and inflammatoryarthropathies. The antagonists may be employed to interfere with thedeleterious cascades attributed primarily to IL-1 and TNF, whichprevents the biosynthesis of other inflammatory cytokines. In this way,the antagonists may be employed to prevent inflammation. The antagonistsmay also be employed to inhibit prostaglandin-independent fever inducedby chemokines. The antagonists may also be employed to treat cases ofbone marrow failure, for example, aplastic anemia and myelodysplasticsyndrome. The antagonists may also be employed to treat asthma andallergy by preventing eosinophil accumulation in the lung. Theantagonists may also be employed to treat subepithelial basementmembrane fibrosis which is a prominent feature of the asthmatic lung.Antibodies against CKα-5 may be employed to bind to and inhibit CKα-5activity to treat ARDS, by preventing infiltration of neutrophils intothe lung after injury. Any of the above antagonists may be employed in acomposition with a pharmaceutically acceptable carrier, e.g., ashereinafter described.

Gene Mapping

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a CKα-5 protein gene. This canbe accomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA then is usedfor in situ chromosome mapping using well known techniques for thispurpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15–25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Fluorescence in situ hybridization (“FISH”) of a cDNA cloneto a metaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance In Man, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1(a) Expression and Purification of “His-tagged” CKα-5E. coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). pQE60 encodes ampicillin antibiotic resistance (“Ampr”) andcontains a bacterial origin of replication (“ori”), an IPTG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that an inserted DNA fragment encodinga polypeptide expresses that polypeptide with the six His residues(i.e., a “6×His tag”) covalently linked to the carboxyl terminus of thatpolypeptide.

The DNA sequence encoding the desired portion CKα-5 protein lacking thehydrophobic leader sequence and transmembrane domain is amplified fromthe deposited cDNA clone using PCR oligonucleotide primers which annealto the amino terminal sequences of the desired portion of the CKα-5protein and to sequences in the deposited construct 3′ to the cDNAcoding sequence. Additional nucleotides containing restriction sites tofacilitate cloning in the pQE60 vector are added to the 5′ and 3′sequences, respectively.

For cloning the mature protein, the 5′ primer has the sequence 5′GCGCCATGGAATGGCAACGAGGGCAGC 3′ (SEQ ID NO:15) containing the underlinedNcoI restriction site. One of ordinary skill in the art wouldappreciate, of course, that the point in the protein coding sequencewhere the 5′ primer begins may be varied to amplify a DNA segmentencoding any desired portion of the complete protein shorter or longerthan the mature form. The 3′ primer has the sequence 5′CGCAAGCTTTTATGTGGCTGATGTCCTGGC 3′ (SEQ ID NO:16) containing theunderlined HindIII restriction.

The amplified CKα-5 DNA fragment and the vector pQE60 are digested withNcoI and HindIII and the digested DNAs are then ligated together.Insertion of the CKα-5 DNA into the restricted pQE60 vector places theCKα-5 protein coding region downstream from the IPTG-inducible promoterand in-frame with an initiating AUG and the six histidine codons.

Alternatively, a preferred bacterial expression vector “pHE4-5”containing an ampicillin resistance gene may be used in this example.pHE4-5/MPIFD23 vector plasmid DNA contains a filler insert betweenunique restriction enzyme sites NdeI and Asp718 and was deposited withthe American Type Culture Collection, 12301 Park Lawn Drive, Rockville,Md. 20852, on Sep. 30, 1997 and given Accession No. 209311. Using 5′ and3′ primers described herein with restriction enzyme sites for NdeI andAsp 718 substituted for the NcoI and HindIII sites in the respectiveprimers, a suitable CKα-5 encoding DNA fragment for subcloning intopHE4-5 can be amplified. The stuffer DNA insert in pHE4-5/MPIFD23 shouldbe removed prior to ligating the CKα-5 fragment to pHE4-5. pHE4-5contains a strong bacterial promoter allowing for high yields of mostheterologous proteins.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),is used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressing CKα-5protein, is available commercially from QIAGEN, Inc., supra.Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3–4 hours at 4° C. in 6M guanidine-HCl,pH 8. The cell debris is removed by centrifugation, and the supernatantcontaining the CKα-5 is loaded onto a nickel-nitrilo-tri-acetic acid(“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra).Proteins with a 6×His tag bind to the Ni-NTA resin with high affinityand can be purified in a simple one-step procedure (for details see: TheQIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the supernatant isloaded onto the column in 6 M guanidine-HCl, pH 8, the column is firstwashed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10volumes of 6 M guanidine-HCl pH 6, and finally the CKα-5 is eluted with6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

The following alternative method may be used to purify CKα-5 expressedin E. coli when it is present in the form of inclusion bodies. Unlessotherwise specified, all of the following steps are conducted at 4–10°C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4–10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells ware then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000–6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2–4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the CKα-5polypeptide-containing supernatant is incubated at 4° C. overnight toallow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded CKα-5 polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 μm membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 mm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

Fractions containing the CKα-5 polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the CKα-5 polypeptide (determined, for instance, by 16%SDS-PAGE) are then pooled.

The resultant CKα-5 polypeptide exhibits greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsare observed from Commassie blue stained 16% SDS-PAGE gel when 5 μg ofpurified protein is loaded. The purified protein is also tested forendotoxin/LPS contamination, and typically the LPS content is less than0.1 ng/ml according to LAL assays.

Example 2 Cloning and Expression of CKα-5 Protein in a BaculovirusExpression System

In this illustrative example, the plasmid shuttle vector pA2 is used toinsert the cloned DNA encoding complete protein, including its naturallyassociated secretory signal (leader) sequence, into a baculovirus toexpress the mature CKα-5 protein, using standard methods as described inSummers et al., A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) followed by convenient restriction sites such as BamHI,Xba I and Asp718. The polyadenylation site of the simian virus 40(“SV40”) is used for efficient polyadenylation. For easy selection ofrecombinant virus, the plasmid contains the beta-galactosidase gene fromE. coli under control of a weak Drosophila promoter in the sameorientation, followed by the polyadenylation signal of the polyhedringene. The inserted genes are flanked on both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31–39 (1989).

The cDNA sequence encoding the full length CKα-5 protein in thedeposited clone, including the AUG initiation codon and the naturallyassociated leader sequence shown in SEQ ID NO:2, is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene. The 5′ primer has the sequence 5′CGCGGATCCGCCATCATGGGACGGGACTTGCGG 3′ (SEQ ID NO:17) containing theunderlined BamHI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947–950 (1987). The 3′ primer has the sequence 5′GCGTCTAGATCAGGTATTAGAGTCAGG 3′ (SEQ ID NO:18) containing the underlinedXbaI restriction site. Those of ordinary skill in the art will recognizethat other primers could be used to generate nucleic acid fragmentsencoding shorter polypeptides. For example, the 5′ primer above (SEQ IDNO:17) with the following 3′ primer could be used to amplify the nucleicacid fragment encoding the soluble extracellular domain:

5′ GCGTCTAGATTATGTGGCTGATGTCCTGGC 3′ (SEQ ID NO:19). The 3′ primercontains the underlined XbaI site.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and XbaI and again ispurified on a 1% agarose gel.

The plasmid is digested with the restriction enzymes BamHI and XbaI andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

Fragment and the dephosphorylated plasmid are ligated together with T4DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria are identified that contain the plasmid with the human CKα-5gene by digesting DNA from individual colonies using BamHI and XbaI andthen analyzing the digestion product by gel electrophoresis. Thesequence of the cloned fragment is confirmed by DNA sequencing. Thisplasmid is designated herein pA2GPCKα-5.

Five μg of the plasmid pA2GPCKα-5 is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413–7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of theplasmid pA2GPCKα-5 are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC® CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate is then incubated for5 hours at 27° C. The transfection solution is then removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum is added. Cultivation is then continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9–10). After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-CKα-5.

To verify the expression of the CKα-5 gene Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus V-CKα-5 at a multiplicity ofinfection (“MOI”) of about 2. If radiolabeled proteins are desired, 6hours later the medium is removed and is replaced with SF900 II mediumminus methionine and cysteine (available from Life Technologies Inc.,Rockville, Md.). After 42 hours, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then are harvested by centrifugation. Theproteins in the supernatant as well as the intracellular proteins areanalyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe mature form or extracellular domain of the CKα-5 protein and thusthe cleavage point and length of the naturally associated secretorysignal peptide.

Example 3 Cloning and Expression of CKα-5 in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC® 37152), pSV2dhfr (ATCC®37146) and pBC12MI (ATCC® 67109). Mammalian host cells that could beused include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 andC127 cells, Cos 1, Cos 7 and CV1, quail QC1–3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, hygromycin allows theidentification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277–279 (1991); Bebbington et al., Bio/Technology 10:169–175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438–447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521–530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

The expression plasmid, pCKα-5HA, is made by cloning a portion of thecDNA encoding the extracellular domain of the CKα-5 protein into theexpression vector pcDNAI/Amp or pcDNAIII (which can be obtained fromInvitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37: 767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

A DNA fragment encoding the soluble extracellular domain of the CKα-5polypeptide is cloned into the polylinker region of the vector so thatrecombinant protein expression is directed by the CMV promoter. Theplasmid construction strategy is as follows. The CKα-5 cDNA of thedeposited clone is amplified using primers that contain convenientrestriction sites, much as described above for construction of vectorsfor expression of CKα-5 in E. coli. Suitable primers include thefollowing, which are used in this example. The 5′ primer, containing theunderlined BamHI site, a Kozak sequence, and an AUG start codon, has thefollowing sequence:

-   5′ CGCGGATCCGCCATCATGGGACGGGACTTGCGG 3′ (SEQ ID NO:17). The 3′    primer, containing the underlined XbaT site has the following    sequence:-   5′ GCGTCTAGATCAGGTATTAGAGTCAGG 3′ (SEQ ID NO:18).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith BamHI and XbaI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the fragment encoding theextracellular domain of the CKα-5 polypeptide.

For expression of recombinant CKα-5, COS cells are transfected with anexpression vector, as described above, using DEAE-DEXTRAN, as described,for instance, in Sambrook et al., Molecular Cloning: a LaboratoryManual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989).Cells are incubated under conditions for expression of CKα-5 by thevector.

Expression of the CKα-5-HA fusion protein is detected by radiolabelingand immunoprecipitation, using methods described in, for example Harlowet al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two daysafter transfection, the cells are labeled by incubation in mediacontaining ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of CKα-5 polypeptide in thisexample. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC®Accession No. 37146). The plasmid contains the mouse DHFR gene undercontrol of the SV40 early promoter. Chinese hamster ovary- or othercells lacking dihydrofolate activity that are transfected with theseplasmids can be selected by growing the cells in a selective medium(alpha minus MEM, Life Technologies) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T.,1978, J. Biol. Chem. 253:1357–1370, Hamlin, J. L. and Ma, C. 1990,Biochem. et Biophys. Acta, 1097:107–143, Page, M. J. and Sydenham, M. A.1991, Biotechnology 9:64–68). Cells grown in increasing concentrationsof MTX develop resistance to the drug by overproducing the targetenzyme, DHFR, as a result of amplification of the DHFR gene. If a secondgene is linked to the DHFR gene, it is usually co-amplified andover-expressed. It is known in the art that this approach may be used todevelop cell lines carrying more than 1,000 copies of the amplifiedgene(s). Subsequently, when the methotrexate is withdrawn, cell linesare obtained which contain the amplified gene integrated into one ormore chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438–447)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521–530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Xba I,and Asp718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman 62-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the CKα-5 polypeptide in a regulated wayin mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad.Sci. USA 89:5547–5551). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzymes BamHI and XbaIand then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

The DNA sequence encoding the extracellular domain of the CKα-5polypeptide is amplified using PCR oligonucleotide primers correspondingto the 5′ and 3′ sequences of the desired portion of the gene. The 5′and 3′ primers are the same as those used in Example 3(a) above.

The amplified fragment is digested with the endonucleases BamHI and XbaIand then purified again on a 1% agarose gel. The isolated fragment andthe dephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10–14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100–200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4 Tissue Distribution of CKα-5 mRNA Expression

Northern blot analysis is carried out to examine CKα-5 gene expressionin human tissues, using methods described by, among others, Sambrook etal., cited above. A cDNA probe containing the entire nucleotide sequenceof the CKα-5 protein (SEQ ID NO:1) is labeled with ³²P using therediprime™ DNA labeling system (Amersham Life Science), according tomanufacturer's instructions. After labeling, the probe is purified usinga CHROMA SPIN-100™ column (Clontech Laboratories, Inc.), according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various human tissues for CKα-5 mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with the labeled probe using ExpressHyb™ hybridizationsolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70° C. overnight, and films developed according tostandard procedures.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

Further, the sequence listing in both hard copy and electronic formsubmitted herewith is incorporated herein by reference.

1. An isolated protein comprising an amino acid sequence selected fromthe group consisting of: (a) amino acid residues 1 to 254 of SEQ IDNO:2; (b) amino acid residues 2 to 254 of SEQ ID NO:2; (c) amino acidresidues 28 to 254 of SEQ ID NO:2; and (d) amino acid residues 28 to 205of SEQ ID NO:2.
 2. The protein of claim 1, wherein the amino acidsequence is (a).
 3. The protein of claim 1, wherein the amino acidsequence is (b).
 4. The protein of claim 1, wherein the amino acidsequence is (c).
 5. The protein of claim 1, wherein the amino acidsequence is (d).
 6. The protein of claim 1 wherein the amino acidsequence further comprises a heterologous polypeptide.
 7. The protein ofclaim 6 wherein the heterologous polypeptide is the Fc domain ofimmunoglobulin.
 8. The protein of claim 1 wherein said protein isglycosylated.
 9. The protein of claim 1 wherein said protein is fused topolyethylene glycol.
 10. An isolated protein produced by a methodcomprising: (a) expressing the protein of claim 1 in a recombinant hostcell comprising a nucleic acid molecule encoding said protein; and (b)recovering the protein from the cell culture.
 11. A compositioncomprising the protein of claim 1 and a carrier.
 12. An isolated proteincomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length polypeptide encoded bythe cDNA clone contained in ATCC® Deposit No. 209231; (b) the amino acidsequence of the full-length polypeptide, excluding the N-terminalmethionine residue, encoded by the cDNA clone contained in ATCC® DepositNo. 209231; (c) the amino acid sequence of the mature polypeptideencoded by the cDNA clone contained in ATCC® Deposit No. 209231; and (d)the amino acid sequence of the extracellular domain of the polypeptideencoded by the cDNA clone contained in ATCC® Deposit No.
 209231. 13. Theprotein of claim 12, wherein the amino acid sequence is (a).
 14. Theprotein of claim 12, wherein the amino acid sequence is (b).
 15. Theprotein of claim 12, wherein the amino acid sequence is (c).
 16. Theprotein of claim 12, wherein the amino acid sequence is (d).
 17. Theprotein of claim 12 wherein the amino acid sequence further comprises aheterologous polypeptide.
 18. The protein of claim 17 wherein theheterologous polypeptide is the Fc domain of immunoglobulin.
 19. Theprotein of claim 12 wherein said protein is glycosylated.
 20. Theprotein of claim 12 wherein said protein is fused to polyethyleneglycol.
 21. An isolated protein produced by a method comprising: (a)expressing the protein of claim 12 in a recombinant host cell comprisinga nucleic acid molecule encoding said protein; and (b) recovering theprotein from the cell culture.
 22. A composition comprising the proteinof claim 12 and a carrier.
 23. An isolated polypeptide consisting of afragment of SEQ ID NO:2, wherein said fragment is at least 30 contiguousamino acid residues in length.
 24. The polypeptide of claim 23 whereinthe fragment is at least 50 contiguous amino acid residues in length.25. The polypeptide of claim 23 fused to a heterologous polypeptide. 26.The polypeptide of claim 25 wherein the heterologous polypeptide is theFc domain of immunoglobulin.
 27. The polypeptide of claim 23 whereinsaid protein is glycosylated.
 28. The polypeptide of claim 23 whereinsaid protein is fused to polyethylene glycol.
 29. An isolatedpolypeptide produced by a method comprising: (a) expressing thepolypeptide of claim 23 in a recombinant host cell comprising a nucleicacid molecule encoding said polypeptide; and (b) recovering thepolypeptide from the cell culture.
 30. A composition comprising thepolypeptide of claim 23 and a carrier.
 31. An isolated polypeptideconsisting of a fragment of the full-length polypeptide encoded by thecDNA clone contained in ATCC® Deposit No. 209231, wherein said fragmentis at least 30 contiguous amino acid residues in length.
 32. Thepolypeptide of claim 31 wherein the fragment is at least 50 contiguousamino acid residues in length.
 33. The polypeptide of claim 31 fused toa heterologous polypeptide.
 34. The polypeptide of claim 33 wherein theheterologous polypeptide is the Fc domain of immunoglobulin.
 35. Thepolypeptide of claim 31 wherein said polypeptide is glycosylated. 36.The polypeptide of claim 31 wherein said polypeptide is fused topolyethylene glycol.
 37. An isolated polypeptide produced by a methodcomprising: (a) expressing the polypeptide of claim 31 in a recombinanthost cell comprising a nucleic acid molecule encoding said polypeptide;and (b) recovering the polypeptide from the cell culture.
 38. Acomposition comprising the polypeptide of claim 31 and a carrier.
 39. Anisolated protein comprising a first amino acid sequence 90% or moreidentical to a second amino acid sequence selected from the groupconsisting of: (a) amino acid residues 1 to 254 of SEQ ID NO:2; (b)amino acid residues 2 to 254 of SEQ ID NO:2; (c) amino acid residues 28to 254 of SEQ ID NO:2; and (d) amino acid residues 28 to 205 of SEQ IDNO:2; wherein said first amino acid sequence is chemotactic for T cells.40. The protein of claim 39 wherein the first amino acid sequence is 90%or more identical to the second amino acid sequence (a).
 41. The proteinof claim 39 wherein the first amino acid sequence is 90% or moreidentical to the second amino acid sequence (b).
 42. The protein ofclaim 39 wherein the first amino acid sequence is 90% or more identicalto the second amino acid sequence (c).
 43. The protein of claim 39wherein the first amino acid sequence is 90% or more identical to thesecond amino acid sequence (d).
 44. The protein of claim 39 wherein thefirst amino acid sequence is 95% or more identical to the second aminoacid sequence (a).
 45. The protein of claim 39 wherein the first aminoacid sequence is 95% or more identical to the second amino acid sequence(b).
 46. The protein of claim 39 wherein the first amino acid sequenceis 95% or more identical to the second amino acid sequence (c).
 47. Theprotein of claim 39 wherein the first amino acid sequence is 95% or moreidentical to the second amino acid sequence (d).
 48. The protein ofclaim 39 wherein the amino acid sequence further comprises aheterologous polypeptide.
 49. The protein of claim 48 wherein theheterologous polypeptide is the Fc domain of immunoglobulin.
 50. Theprotein of claim 39 wherein said protein is glycosylated.
 51. Theprotein of claim 39 wherein said protein is fused to polyethyleneglycol.
 52. An isolated protein produced by a method comprising: (a)expressing the protein of claim 39 in a recombinant host cell comprisinga nucleic acid molecule encoding said protein; and (b) recovering theprotein from the cell culture.
 53. A composition comprising the proteinof claim 39 and a carrier.
 54. An isolated protein comprising a firstamino acid sequence 90% or more identical to a second amino acidsequence selected from the group consisting of: (a) the amino acidsequence of the full-length polypeptide encoded by the cDNA clonecontained in ATCC® Deposit No. 209231; (b) the amino acid sequence ofthe full-length polypeptide, excluding the N-terminal methionineresidue, encoded by the cDNA clone contained in ATCC® Deposit No.209231; (c) the amino acid sequence of the mature polypeptide encoded bythe cDNA clone contained in ATCC® Deposit No. 209231; and (d) the aminoacid sequence of the extracellular domain of the polypeptide encoded bythe cDNA clone contained in ATCC® Deposit No. 209231; wherein said firstamino acid sequence is chemotactic for T cells.
 55. The protein of claim54 wherein the first amino acid sequence is 90% or more identical to thesecond amino acid sequence (a).
 56. The protein of claim 54 wherein thefirst amino acid sequence is 90% or more identical to the second aminoacid sequence (b).
 57. The protein of claim 54 wherein the first aminoacid sequence is 90% or more identical to the second amino acid sequence(c).
 58. The protein of claim 54 wherein the first amino acid sequenceis 90% or more identical to the second amino acid sequence (d).
 59. Theprotein of claim 54 wherein the first amino acid sequence is 95% or moreidentical to the second amino acid sequence (a).
 60. The protein ofclaim 54 wherein the first amino acid sequence is 95% or more identicalto the second amino acid sequence (b).
 61. The protein of claim 54wherein the first amino acid sequence is 95% or more identical to thesecond amino acid sequence (c).
 62. The protein of claim 54 wherein thefirst amino acid sequence is 95% or more identical to the second aminoacid sequence (d).
 63. The protein of claim 54 wherein the amino acidsequence further comprises a heterologous polypeptide.
 64. The proteinof claim 63 wherein the heterologous polypeptide is the Fc domain ofimmunoglobulin.
 65. The protein of claim 54 wherein said protein isglycosylated.
 66. The protein of claim 54 wherein said protein is fusedto polyethylene glycol.
 67. An isolated protein produced by a methodcomprising: (a) expressing the protein of claim 54 in a recombinant hostcell comprising a nucleic acid molecule encoding said protein; and (b)recovering the protein from the cell culture.
 68. A compositioncomprising the protein of claim 54 and a carrier.
 69. An isolatedpolypeptide consisting of a fragment of the polypeptide of SEQ ID NO:2wherein said fragment is chemotactic for T cells.
 70. The polypeptide ofclaim 69 wherein the polypeptide is fused to a heterologous polypeptide.71. The polypeptide of claim 70 wherein the heterologous polypeptide isthe Fc domain of immunoglobulin.
 72. The polypeptide of claim 69 whereinsaid protein is glycosylated.
 73. The polypeptide of claim 69 whereinsaid polypeptide is fused to polyethylene glycol.
 74. An isolatedpolypeptide produced by a method comprising: (a) expressing thepolypeptide of claim 69 in a recombinant host cell comprising a nucleicacid molecule encoding said polypeptide; and (b) recovering thepolypeptide from the cell culture.
 75. A composition comprising thepolypeptide of claim 69 and a carrier.